This invention relates to a new fusion protein, its cDNA, and a vector and a cell comprising said cDNA. Furthermore, this invention relates to the use of said fusion protein in an immunoassay for simultaneous detection of autoantibodies related to insulin dependent diabetes mellitus.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
GAD65, IA2 and insulin are pancreatic proteins produced by the beta cells (for review see Atkinson and Maclaren 1993). Autoantibodies to these proteins are detected in patients with insulin-dependent diabetes mellitus (IDDM) and healthy individuals at risk for developing the disease. More than 80% of newly-diagnosed IDDM patients have antibodies against at least one of these proteins (Baekkeskov et al. 1982). The risk of diabetes in relatives of IDDM patients increases markedly when the number of autoantibodies detected in the serum increases (Bingley et al. 1994; Verge et al. 1994). In a group of high genetic risk, presence in serum of antibodies to one or more of these autoantigens predicted the disease onset accurately (Verge et al. 1996). Also permanently healthy subjects (as regards IDDM) may have temporarily or permanently antibodies against one of the three antigens, but antibodies against multiple antigens occur extremely rarely. It is therefore sought to simultaneously determine reactivity against two or all three of the proteins, as the positivity for more than one of these autoantibodies remarkably increases disease risk (Bingley et al. 1994).
GAD65 (Bu et al. 1992) has several epitopes recognised by autoantibodies (Falorni et al. 1996). These are located mostly at the center and C-terminus of the molecule whereas the N-terminal quarter of the molecule is thought to contribute to membrane docking of the protein, and to contain few if any IDDM-informative epitopes (Falorni et al. 1996).
IA2 (also known as ICA512) (Rabin et al. 1994) is a transmembrane protein with still unknown function. The intracellular part of the molecule (IA2ic, about 40 kDa) contains a domain with similarity to the active center of protein phosphatases (Fischer et al. 1991), but no enzymatic activity has been ascribed the IA2 molecule. The informative epitopes of IA2 reside in the cytoplasmic domain and herein they are concentrated at the C-terminal half (Lampasona et al. 1996; Zhang et al. 1997).
Insulin (Bell et al. 1980) is made by pancreatic xcex2-cells as a precursor preproinsulin which is cleaved to proinsulin. The proinsulin is further processed to give the insulin consisting of A and B chains connected together with two disulphide bridges.
More than 20% of sera collected from newly-diagnosed IDDM-patients contain insulin autoantibodies (IAA) (Sabbah et al. 1996). As, however, the immunity to insulin may have arisen through formation of response to prepro- or proinsulins (Snorgaard et al. 1996), it is relevant to use these peptides in this assay system. Tolerance to this autoantigen may be induced by oral insulin feeding in non-obese diabetic (NOD) mice (Zhang et al. 1991).
In addition to linear epitopes, autoantibodies are thought to recognize important conformational epitopes resulting from the three-dimensional structure of the protein (Kim et al. 1993). Antigen molecules produced or assayed using techniques which destroy these structures are less informative as regards IDDM or prediabetes.
Several methods for detection of autoantibodies in IDDM sera have been elaborated. One method exploits in vitro transcription-translation for producing radioactively labeled autoantigen (IA2, GAD65) (Petersen et al. 1994), while in another method biotin-labeled GAD65 is added to the patient sera and after formation of immune complexes, free label is detected and quantitated (Mehta et al. 1996). These methods all suffer from suboptimal niveau of informativity, as they employ only one specific autoantigen. Moreover they have the drawbacks associated with the use of radiochemicals.
Using a protein molecule in which a combination of the epitopes from at least two but preferably three different autoantigens are represented should detect a larger panel of autoantibodies thus revealing more specifically the population of individuals at risk of developing the disease.
According to one aspect, this invention relates to a new fusion protein having epitopes of at least two of the autoantigens glutamic acid decarboxylase (GAD65), islet cell antigen (IA2) and preproinsulin (PPINS) wherein said epitopes are connected with a linker peptide, said fusion protein being able to bind to a solid phase.
According to another aspect, the invention concerns a cDNA sequence encoding the said fusion protein.
According to a third aspect, the invention concerns a vector and a cell comprising said cDNA.
According to a fourth aspect, the invention concerns an immunoassay for the simultaneous determination in a sample of a person""s body fluid of at least two insulin-dependent diabetes mellitus (IDDM)-related autoantibodies, wherein each autoantibody is specific for an epitope of the autoantigens glutamic acid decarboxylase (GAD65), islet cell antigen (IA2) or preproinsulin (PPINS). The immunoassay comprises the steps of
incubating said sample with said autoantigens or, alternatively, with the fusion protein according to this invention, said autoantigens or said fusion protein being bound to a solid support,
adding at least one labeled reagent capable of binding to one or more of said autoantibodies, and
quantifying the signals from the labels bound to the solid phase.
According to still one aspect, the invention concerns a method for diagnosing a person""s risk of developing insulin-dependent diabetes mellitus (IDDM), said method comprising the determination in a sample of said person""s body fluid of at least two insulin dependent diabetes mellitus (IDDM)-related autoantibodies specific for an epitope of the autoantigens glutamic acid decarboxylase (GAD65), islet cell antigen (IA2) or preproinsulin (PPINS), wherein the presence of at least two of said autoantibodies are indicative for said person""s risk of developing IDDM. The order of appearance of these autoantibodies is used to predict the time point of onset of the disease.